We seek renewal of a highly productive multiple-PI research program involving the analysis and engineering of a broad class of monooxygenases from diverse natural product pathways. Cytochrome P450s are one of the most widely distributed groups of enzymes in nature, catalyzing the oxidation of natural product and xenobiotic small molecules. Although hundreds of P450s have been examined in the oxidative metabolism of xenobiotics and steroids, only a small number have been studied in bacterial secondary metabolism, especially in macrolide antibiotic biosynthetic pathways. In most of these systems, hydroxylation and/or epoxidation reactions occur in the late stages of biosynthesis after macrolide formation by the polyketide synthase (PKS). In addition to significant increases in biological potency, hydroxylation provides potential sites for chemical modification and further enhancement of bioactivities. Thus, the creation of novel macrolide analogs through in vivo metabolic engineering and in vitro chemoenzymatic synthesis warrants a concomitant effort towards the development of monooxygenases with defined substrate specificities. The aim of the proposed work is to expand our understanding of the substrate flexibility and functionality of a range of P450 monooxygenases from macrolide and select other natural product systems. Our progress over the first period of support has provided fascinating new insights into the molecular mechanisms of these biocatalysts, and their ability to generate novel products by hydroxylation, and epoxidation of natural and unnatural substrates. This information will direct protein engineering/substrate engineering efforts to better understand the function and positional specificity of the enzyme, as well as its ability to catalyze a range of oxidative reactions. Our program brings complementary approaches of synthetic chemistry to create diverse substrates, biochemistry to investigate and develop engineered monoxygenases with versatile substrate selectivity, and X-ray and NMR- based methods to obtain high resolution structural information for mechanistic understanding of these remarkable proteins. Specific Aim 1. Assess the impact of steric, electronic and directing group factors on catalytic promiscuity in the P450 PikC using a series of synthetic analogs of the natural macrolide substrate YC-17. Employ X-ray and solution NMR based structural biology approaches to gain detailed insights into binding parameters, protein-substrate dynamics, and the mechanistic basis for regio- and stereochemical specificity of natural and unnatural substrates. Specific Aim 2. Expand access to diverse synthetic substrates for a range of new P450 enzymes to investigate regio- and stereochemical details of monooxygenase-catalyzed hydroxylation and epoxidation reactions. Specific Aim 3. Pursue biochemical and structural studies of mixed-function iterative P450 enzymes to analyze substrate specificity and kinetics, as well as to investigate binding and catalytic mechanisms.